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NEUROGENETIC ANALYSIS OF HEREDITARY NEUROPATHIES IN THE ERA OF GENOMIC

MEDICINE

Ph.D. thesis

György Máté Milley M.D.

Neuroscience Doctoral School Semmelweis University

Supervisor: Anikó Gál Ph.D.

Official reviewers: Dóra Nagy Ph.D.

Eszter Jávorszky Ph.D.

Head of the Final Examination Committee: Miklós Szabó M.D. Ph.D.

Members of the Final Examination Committee: Bence Gunda MD. Ph.D.

György Báthori MD. Ph.D.

Budapest

2019

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2 Table of Content

The list of Abbreviations ... 4

1. Introduction ... 9

1.1 Historical overview ... 10

1.2 Classification ... 12

1.3 Symptoms and signs ... 13

1.4 Genetic background ... 14

1.4.1 Frequent CMT genes ... 17

1.4.2 Less frequent CMT genes ... 22

1.5 Evaluation of the hereditary neuropathies ... 23

1.5.1 Clinical assessment ... 23

1.5.2 Nerve conduction study ... 26

1.5.3 Nerve biopsy and imaging studies ... 27

1.5.4 Genetic diagnostics ... 28

1.6 Management of hereditary neuropathies ... 29

1.6.1 Genetic counseling ... 29

1.6.2 Therapy and treatment ... 30

1.6.3 Avoid of medications ... 31

1.6.4 CMT and pregnancy ... 31

2. Objectives ... 32

3. Methods ... 33

3.1 Clinical and electrophysiological characterization of the cohort studied ... 33

3.2 Genetic testing ... 34

3.3 In silico, pathogenicity and statistical analyzes ... 36

4. Results ... 37

4.1 Clinical and electrophysiological assessment ... 37

4.2 Genetic testing and distribution of genetic subtypes ... 37

4.3 Novel alterations in our CMT patients ... 39

4.3.1 Clinical description of patients with novel alterations ... 40

4.4 Investigation of the phenotypic spectrum in CMT subtypes ... 46

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4.5 Clinical features and gender comparison of a set of patients carrying GJB1

mutation ... 49

4.6 Analysis of rare variants with high-througput methodology. ... 53

4.6.1 TRPV4 pathogenic alterations ... 53

4.6.2 POLG and MME likely pathogenic alterations ... 54

4.6.3 HINT1 pathogenic variant ... 55

5. Discussion ... 57

5.1 Frequency of CMT genes in Hungary ... 57

5.2 Phenotypic spectrum of CMT genes in Hungarian patients ... 58

5.3 Clinical and electrophysiological analysis of a set of CMTX1 patients ... 59

5.4 Rare variants identified with high-throughput methods ... 61

6. Conclusions ... 64

7. Összefoglaló ... 66

8. Summary ... 67

9. Bibliography ... 68

10. Bibliography of the candidate’s publications ... 91

11. Acknowledgements ... 93

12. Supplement ... 94

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The list of Abbreviations

ACMG – American College od Medical Genetics and Genomics ADOA – Autosomal dominant optic atrophy

ALS – Amyotrophic lateral sclerosis ANA – Anti-nuclear antibody

ANCA – Anti-neutrophil cytoplasmic antibodies ANS – Autonomic nervous system

ax – Axonal

CCFDN – Congenital cataracts, facial dysmorphism, and neuropathy CDSMA – Dominant congenital spinal muscular atrophy

CHN – Congenital hereditary neuropathy CI95% - 95% confidence interval

CIDP – Chronic inflammatory demyelinating polyradiculoneuropathy CK – Creatinine kinase

CMAP – Compound muscle action potential CMT–Charcot-Marie-Tooth disease

CMTES – Charcot-Marie-Tooth examination score CMTNS – Charcot-Marie-Tooth neuropathy score CNS – Central nervous system

CNV – Copy number variant CSA – Cross-sectional area CSF – Corticospinal fluid

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CTDP1 – C-terminal domain of RNA polymerase II subunit A Cx32 – Connexin 32

de – Demyelinating

dHSN – Distal hereditary motor neuropathy

DI-CMT – Dominant intermediate Charcot-Marie-Tooth disease DNA – Deoxyribonucleic acid

DSS – Déjerine-Sottas syndrome EGR2 – Early growth factor 2 EM – Electron microscopy EMG – Electromyopgraphy ENG – Electroneurography ENMG – Electroneuromyography

FSGS – Focal segmental glomerulosclerosis

GDAP1 – Ganglioside-induced differentiation-associated protein 1 GJB1 – Gap junction beta 1

GTF – General transcription factor HBV – Hepaitis B virus

HCV – Hepatitis C virus

HIV – Human immunodeficiency virus

HMSN – Hereditary sensory and motor neuropathy HNPP – Hereditary neuropathy with pressure palsy HR1/2 – Helical hapted repeat regions

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HSAN – Hereditary sensory and autonomic neuropathy HSN – Hereditary Sensory Neuropathy

HSP – Hereditary spastic paraplegia in – Intermediate

INF2 – Inverted Formin 2

IVIg – Intravenous immunoglobulins LDH – Lactate dehydrogenase

MADSAM – Multifocal acquired demyelinating sensory and motor neuropathy MAG – Myelin-associated glycoprotein precursor

MFN2 – Mitofusin 2

MGUS – Monoclonal gammopathy with uncertain significance MIRAS – Mitochondrial recessive ataxia syndrome

MLPA – Multiplex ligand probe assay MME – Membrane metalloendopeptidase MMN – Multifocal motor neuropathy MNCV – Mean nerve conduction velocity MRC – Medical Research Council

MRI – Magnetic resonance imaging

mtDNA – Mitochondrial deoxyribonucleic acid NADH – Nicotinamid adenine dinucleotide NCS – Nerve conduction study

NCV – Nerve conduction velocity

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7 nDNA – Nuclear deoxyribonucleic acid NDRG1 - N-myc downstream regulated 1 NEFL – Neurofilament L

NGS – New generation sequencing NMD – Neuromuscular disorder OR – Odds ratio

p.s. – Present study

P/F – Patient per family ratio P0/MPZ – Myelin protein zero PCR – Polymerase chain reaction

PEO – Progressive external ophtalmoplegia PMP22 – Peripheral myelin protein 22 PNS – Peripheral nervous system

POLR2A – RNA polymerase 2 subunit A qPCR – Quantitative polymerase chain reaction RFLP – Restricted fragment length polymorphism RLS – Roussy-Lévy syndrome

RNA – ribonucleic acid

SANDO – Sensory ataxia neuropathy dysarthria and opthalmoplegia SAP – Sensory action potential

SLE – systematic lupus erythematosus SMA – Spinal muscular atrophy

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8 SMN1 – Survival of motor neuron 1

SNP – Singe nucleotide polymorphism SNV – singe nucleotide variant

SPSMA – Scapuloperoneal spinal muscular atrophy

SPTLC1/2 – Serine palmitoyltransferase, long chain base subunit 1/2 TM1/2 – Transmembrane anchor domain ½

TRPV4 – Transient receptor potential cation channel subfamily V member 4 TSH – Thyroid stimulating hormone

UMN – Upper motor neuoron

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1. Introduction

Peripheral neuropathy is a term for a group of conditions in which the peripheral nerves are damaged. Nerve damage can impair the muscle strength, the sensation, and different organ functions. In general, peripheral neuropathies can be classified according to the (i) number of affected nerves – mononeuropathy (one), multifocal neuropathy (multiple) or polyneuropathy (numerous nerves); (ii) pattern of impairment – symmetric or asymmetric (iii) type of involved nerves – motor, sensory or both types of nerves; (iv) type of nerve lesion – demyelinating, axonal or mixed, and (v) course of neuropathy – acute or chronic condition (1).

Numerous nerves are damaged in polyneuropathy at the same time resulting in broad spectrum of clinical symptoms. Length-dependent nerve degeneration causes the first symptoms in limbs, spreading proximal and worsening progressively. Common causes of polyneuropathy are (i) diabetes, (ii) alcohol abuse, (iii) infection or (iv) drug related nerve damage while other possible etiology with a lower incidence can be (v) tumors, (vi) metabolic and (vii) autoimmune diseases or (viii) hereditary disorders (1).

Hereditary neuropathies are chronic conditions affecting symmetric the motor and/or sensory nerves. It is one of the most common inherited neurodegenerative disorders, affecting approximately every one person from 2500 (2). The relative homogenous clinical appearance of the disease is associated with an especially wide genetic background. Charcot-Marie-Tooth disease is the eponym of hereditary motor and sensory neuropathy but related disorders – hereditary distal motor neuropathy (dHMN) and hereditary sensory neuropathy (HSN) – are also considered as subgroups of CMT (3, 4).

The expanding knowledge about hereditary neuropathies has broken with the previous conventions and indicated more recent perspectives in the last two and half decades.

Because of the extensive size of the topic and the scarce extent of the space, figures, flow charts, and tables are welcomed to use for detailed and easily understandable demonstration of topics.

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10 1.1 Historical overview

The first descriptions of the inherited distal muscle weakness and wasting, calling it peroneal muscular atrophy, have been reported by Jean Martin Charcot with Pierre Marie and by Howard Henry Tooth in 1886, separately (5). Later, Johann Hoffman described a histological finding of thickened nerves in a case with peroneal muscular atrophy in 1912 (6). From this point, the disorder is also referred as Charcot-Marie-Tooth-Hoffmann disease.

In 1888, Herringham, ahead of his time, has observed a family in which only males were affected supposing a distinct genetic feature between males and females (7). In the following century, the existence of X-linked inherited neuropathy has been questioned by Harding (8); however, it has been later ascertained that the X-linked dominant form of CMT (CMTX1) is the second most common genetic cause of hereditary sensorimotor neuropathies. Eponyms, like Roussy-Lévy syndrome (RLS) and Déjerine-Sottas syndrome (DSS), has become obsolete in the last decades as well, after the genetic analyses have proved the same genetic background with CMT (9-13). In spite of that, Hereditary Neuropathy with Pressure Palsy (HNPP) has a distinct genetic cause and different clinical features than CMT so HNPP is still considered as an independent entity among hereditary motor and sensory neuropathy (HMSN) (14).

Nerve biopsy and pathological diagnosis meant to be the main diagnostic approach in the early decades of the 20th Century. Since the late 1970s, electroneuromyography (ENMG) has become a ubiquitous diagnostic tool providing new frontiers of non-invasive diagnosis, classification and efficient follow-up of hereditary and other neuropathies (15).

Recently, nerve ultrasound and magnetic resonance imaging seem to be a new trend to assign the need for genetic testing, further improving the diagnostic algorithm of neuropathies and also proved their reliability in follow-up studies (5).

The genetic era has officially begun in 1991 with the identification of the first disease causing genetic variant in the PMP22 gene (16). Since that time, the research has been accelerated and it is still at a bold pinnacle of discoveries of novel genes and genomic mechanisms [Fig. 1]. Until now, more than 90 genes have been related to hereditary neuropathies, however, there is a remarkable hiatus in the list of genes. The extended use

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of next generation sequencing started in 2009 which provided a decent catalyst for novel findings (4).

Fig. 1 A: The diagram indicates the extension of the literature related to Charcot-Marie- Tooth neuropathies in the previous decades. The number of publication (y axis) was determined by using the PubMed database (http://www.ncbi.nlm.nih.gov/pubmed/). Data are visualized in five years divisions from 1981 to 2015 and they are arranged in chronological order (x axis). B: The diagram shows the number of discovered genes per year related to hereditary neuropathy (y axis) since 1991. The number of genes is showed per year arranged in decreasing order (x axis). Data are based on supplement 1.

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12 1.2 Classification

The classification of hereditary neuropathies had undergone numerous changes depending on the prevailing views about the disease. The first subsets were based on the clinical description of the disease, usually used their eponyms for naming them. The expanding knowledge and the novel diagnostic possibilities have later required new approaches leading to further reconsideration of the actuel classificiations. Nowadays, the genetic classification is reckoned as the most sophisticated way of categorization but this detailed subdivision is quite a challenge in the daily practice (1) [Table 1, Suppl. 1].

Table 1. The different classifications of hereditary motor and sensory neuropathies since the first descriptions. Notes: a: it has been first introduced by Dyck and revised by Thomas and Harding in 1980 (17); b: The nerve conduction velocity (NCV) of the intermediate form is also referred to 30-40 m/s. Mixed pathological findings should be also a cirterion (18).

Original descriptions of inherited neuropathies

According to the clinical and pathological features along with the

inheritance pattern (from 1968) a

According to the nerve conduction studies (from 1980)

Genetic classification

(from 1997)

Charcot-Marie-Tooth- (Hoffmann) disease (peroneal muscular atrophy)

HMSN type I

(autosomal-dominant form with low conduction velocities and segmental demyelination and remyelination)

CMT1 (demyelinating form,

NCV <38m/s)

CMT1 autosomal

dominant myelinopathy Déjerine-Sottas disease

(autosomal recessive, severe and early onset hypertrophic neuropathy with CSF protein elevation)

HMSN type II (autosomal dominant form with normal nerve conduction velocities and amplitude-reduction while nerve

pathology shows axonal features)

CMT2 (axonal form NCV >38m/s)

CMT2 autosomal dominant and

recessive axonopathy Roussy-Lévy syndrome

(inherited hypertrophic neuropathy and tremor)

HMSN type III (Déjèrine-Sottas disease)

CMTIb (intermediate form)

NCV 25-45m/s

DI-CMT intermediate form Congenital

Hypomyelinating Neuropathy (neonatal hypotonia)

HMSN type IV (autosomal recessive form of hereditary motor and sensory

neuropathies)

HSAN and dHMN (depending on the severity of motor or sensory deficiency)

CMT4 either myelinopathy or

axonopathy, recessive forms Hereditary

Neuropathy with Pressure Palsy (focal neuropathy in

predilection spots)

HMSN type V

(HMSN and spastic paraplegia are simultaneously present)

CMTX X-linked inheritance HMSN type VI and VII

(HMSN with optic atrophy and/or pigmentary retinopathy)

dHMN overlapping forms

with HMSN dHMN or HSANd

(depending on the severity of motor or sensory deficiency)

HSAN overlapping forms

with HMSN

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In addition, the genetic classification of CMT2 has reached and overcame the physical barriers of alphabets, therefore, its modification or expansion needs to be considered as well.

All the classification are presently used in different combinations to describe the disease accurately. Determining of genetic etiology is not always possible and genetic testing needs to be designed carefully. Prior the testing, multiple factors should be taken into account: (i) patient’s phenotype (ii) country-specific genetic epidemiology data, (iii) availability of genetic screening, (iv) methodology, (v) aim of genetic testing (differential diagnostics, preconception, presymptomatic or prenatal counseling, therapy etc.) (1, 4).

1.3 Symptoms and signs

Charcot-Marie-Tooth neuropathy affects fundamentally both the motor and sensory nerves. It is characterized by length dependent nerve degeneration which slowly progresses with time leading to worsening of the condition and a gradually developing disability (19). It starts usually in the first three decades of life (20).

The symptoms of classical phenotype occur primarily in the areas of the nerve damage.

Classical phenotype is characterized by distal muscle weakness (impaired tip toe and heel walking, paretic gait, difficulty using zippers and buttons as well as clumsiness in manipulating small objects), muscle atrophy (inverted champagne bottle appearance due to the loss of muscle bulk), hypotonia, reduction or abolition of tendon reflexes, distal sensory loss (e.g. paresthesia, hypoesthesia, anesthesia, sensory ataxia or pain) and often associated with fasciculation, cramps or orthopedic deformities, such as hollow feet, foot drop, scoliosis etc. [Fig. 2] (1, 19). The disease leads slowly and gradually to impaired mobility in most patients but they remain ambulatory supported by orthesis ofirst threer walking cane while wheelchair-dependency is infrequent and become required generally after the fourth decade of life (21). Pupils may have problems with handwriting in schools (22).

Associated features occur frequently due to the complex physiological roles of different CMT genes resulting in atypical signs and symptoms and leading to difficulties in distinguishing CMT from other disorders (see chapter 2.4.). If atypical clinical signs and

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symptoms are present, it can inflict overlapping phenotypes with spinal muscular atrophy (SMA), hereditary spastic paraplegy (HSP), amyotrophic lateral sclerosis (ALS), autosomal dominant optic atrophy (ADOA), ataxias or mitochondrial disorders (4).

Overlapping phenotypes lead to overlapping pharmacotherapies which further emphasize the importance of genetic profiling in the near and distant future.

Fig. 2 27 -year-old male patient harboring the entire deletion of the coding region of GJB1 gene. The patient above has moderate sensomotoric neuropathy with a CMTNS of 11/36. The photos were taken by the authors. Patient has consented to take the photographs and to press them.

A: inverted champagne bottle appearance due to the loss of muscle bulk while proximal musculature is spared; B: pes cavus and atrophy of the small foot muscles;

C: atrophy of the small hand muscles and deformation of the fingers.

1.4 Genetic background

Until now, more than 90 genes are known to be related to Charcot-Marie-Tooth disease [Suppl. 1]. The proteins, encoded by CMT genes, are involved in many different

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physiological functions of neuronal cells, i.e. (i) myelin proteins; (ii) cytoskeleton, nucleoskeleton and nuclear envelope; (iii) transcriptional regulation (iv) protein biosynthesis; (v) protein modification, folding and degradation (vi) intracellular transport;

(vii) ER membrane shaping; (viii) mitochondrial dynamics; (ix) mitochondrial energy production; (x) sphingolipid biosynthesis; (xi) phosphoinositide metabolism; (xii) Rho GTPase signaling; (xiii) interaction with the extracellular environment (xiv) ion channels and (xv) others [Fig. 3] (23).

Fig. 3 Functions of proteins encoded by different CMT associated genes (23).

Depending on the causative CMT gene, inheritance can be autosomal dominant or recessive, or X-linked dominant or recessive. Interestingly, few genes show recessive and dominant inheritance, as well (19). Different genes cause a different type of neuropathies while the clinical presentation can further vary the overall picture (see par. 2.2 and 2.3) [Fig. 4].

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Fig. 4 The figure demonstrates the overlap of clinical and electrophysiological features along with the genetic classification of HMSN without a claim to completeness. It is worth to note that some of the genes (MFN2, MPZ, HSPB1, EGR2 etc.) can be categorized differently which further complicate the classification (5).

The four most frequent causative genes are PMP22, GJB1, MPZ and MFN2 in order and these are responsible for 70-90 percent of all CMT cases (24). The frequency of major and minor genes varies widely in different cohorts and because of the founder effect, some of the genetic variants are mainly specific for a certain ethnicity or geographical area. Because of the high number of CMT genes, this paper is not suitable for a detailed enumeration of genes, therefore, we discuss here the actually studied part of CMT genetics (2.4.1. and 2.4.2). Further genes are collected in the supplement (Suppl. 1).

The expanding number of clinical data have proved the heterogeneity of the appearance of CMT. Natural histories of patients varies greatly even in families with same mutation.

This is especially true in GJB1 families where females tend to have milder phenotypes than males [24]. High number of potential modifying factors, such as gene-gene interactions, mutation load, epigenetic modification effects, co-morbidities or disease management, likely have an impact on its characteristics and leads to a less predictable disease progression (25-29). Recently, extensive genotype-phenotype meta-analyses studied the possible correlation between various symptoms, disease severity and genes (30, 31). It is well known that progression of neuropathy is linked to age but its dynamics and age of onset vary widely. It worth to note that around seventy percent of CMT starts before the third decade of life (32).

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Depending on the causative gene, additional features can occur in CMT as well. Signs of chronic pyramid tract involvement, spasticity, nystagmus or ataxia are sensitive markers for MFN2 and GJB1 mutations (33, 34). Hearing impairment is a common feature in PMP22, GJB1, MPZ, NDRG1 neuropathies (35-38). Genes, which are involved in mitochondrial dynamics (MFN2, GDAP1, DNM2), frequently cause mtDNA deletion or depletion, optic atrophy or cerebellar ataxia (39-42) while myopathy might be present with DNM2 or LMNA mutation (40). The connection between PMP22 duplication and immunological abnormalities has been also reported multiple occasions (43, 44).

Proteinuria, FSGS, and deafness occur together in INF2 mutations (45, 46). Furthermore, respiratory difficulties, phonation disturbances, and age at onset can be related to certain genotypes as well. For a detailed collection of reported associated features see Suppl. 1.

1.4.1 Frequent CMT genes

1.4.1.1 Peripheral Myelin Protein 22 (PMP22) MIM #601097

Cytogenetic location of PMP22 is on 17p12 chromosome region (MIM #601097).

PMP22 encodes a myelin structure protein which comprises 2 to 5% of myelin sheet in the PNS (47). Although mainly expressed in PNS, PMP22 mRNA expression has been also found in CNS. Studies also suggest a potential role in nerve regeneration, Schwann cell differentiation, and growth (48).

In 1991, PMP22 duplication was the first mutation identified in CMT and later its deletion was linked to HNPP in 1993 (14). PMP22 gene is functionally not disrupted in eithers so gene-dosage effect seems to be the potential explanation for the disorder. Reiter et al identified the molecular etiology where they observed the homologous recombination event that was responsible for the unequal crossing over causing a deletion and a duplication at the same time in two sister chromatids (49).

PMP22 duplication and point mutations have been linked to CMT1A and CMT1E, respectively. CMT1A is the most common cause of hereditary neuropathies (35-45%) whereas CMT1E incidence seems to be less prominent (<1%) (50, 30, 51). Symptoms, severity, and age of onset in CMT1A vary widely. Molecular genetic analysis has linked the gene to Dejerine-Sottas syndrome and Roussy-Levy syndrome as well which should be considered as part of the CMT1A spectrum (MIM #601097). Frequently, the disease

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starts in a couple of years after birth but some cases can be completely symptomless during entire life. CMT1A is characterized by classic symptoms of demyelinating neuropathy but may occur along with tremor, cranial nerve involvement – facial weakness, hearing impairment, and vocal cord palsy – or autonomic dysfunction, too.

CNS may also be affected by nystagmus, pyramidal signs, white and grey matter volume reduction or white matter lesions (31). An elevated level of PMP22 protein may trigger the immune dysfunction since CMT1A occasionally associate with chronic inflammatory demyelinating polyneuropathy (CIDP) and other autoimmune diseases (52).

1.4.1.2 Gap Junction Protein Beta 1 (GJB1)

Gap junction beta 1 (GJB1) gene is located in Xq13.1 chromosome region and encodes Connexin 32 (Cx32) proteins. GJB1 is a member of connexin protein family (53) which form gap junction channels and are involved in the transport of small molecular weight substances (<1kDa). A connexin protein contains four transmembrane (M1-M4), two extracellular (E1-2), and one cytoplasmic loop domain (IC) [Fig. 5/A]. Six of same connexins form a connexon also called hemichannel [Fig. 5/C] which pairs with the connexons of adjacent membrane creating gap junction channels [Fig. 5/B]. Due to the highly homologous connexin proteins, different connexons can form heterodimer pores as well (54) (55).

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Fig. 5 (A) Structure of connexin in the membrane. © refers to cysteine rich regions of extracellular domains. (B) Hemichannels of adjacent membranes create the gap junction channel. (C) The structure of connexon is formed by six homolog connexin molecules where the pore in the middle is gathered around by them. Pores can be both opened or closed state influenced by current voltage (54).

Cx32 is localized in many different cell types including central and peripheral nerves, hepatocytes, pancreatic and embryonal cells (56). In peripheral nerves, Cx32 is located in the paranodal region and the Schmidt-Lantermann incisures of Schwann cells. It has a crucial role in maintaining normal myelination in the peripheral nervous system (57, 58).

Pathogenic mutations of GJB1 cause X-linked dominant form (CMTX1). To date, more than 400 GJB1 pathogenic variants have been identified as the cause of CMTX1 including CNVs. CMT1X is responsible for 8.3 to 12.8 percent of all CMT cases, and after PMP22 gene duplication, it is the second most common cause of CMT (59). CMTX1 shows a wide spectrum of sensory and motor symptoms, and the nerve conduction studies reveal all forms of sensorimotor neuropathy (60). Though CMTX1 is considered to show X- dominant inheritance, female carriers usually show milder clinical symptoms than males with the same genotype (61, 62). Certain GJB1 pathogenic alterations were in association with central nervous system involvement and sensorineural hearing loss as well (63-65).

Most of the GJB1 mutations cause CMT through loss of normal connexin function but it

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is also suspected that the mutant protein has a dominant negative effect and suppresses the function of other gap junction proteins which would be able to replace them (66, 67).

Presumably, both pathogenic changes are possible but depending on certain variants (68).

1.4.1.3 Myelin Protein Zero (MPZ, P0)

Myelin protein zero (MPZ) gene is located on 1q23.3 chromosome and encodes the major structural protein of peripheral myelin sheet and accounts for more than 50% of myelin proteins. It expresses only in PNS (69). MPZ mutations may refer to the third most frequent genetic cause of CMT, it accounts for 3-7% of all cases (50, 30, 51).

In 1993, Hayasaka and al identified MPZ as the cause of CMT1B (70). MPZ pathogenic alterations can cause a wide spectrum of hereditary neuropathy – it was found in association with Dejerinne-Sottas syndrome, Roussy-Lévy syndrome, congenital hypomyelinating neuropathy (71). Electrophysiological patterns vary as well, demyelinating, axonal or intermediate types can be also recorded with ENG. Cranial nerve involvement, pupil abnormalities, skeletal deformities, deafness, cognitive impairment, CNS involvement may be associated features, too (31).

1.4.1.4 Mitofusin2 (MFN2)

MFN2 is located on 1p36.2 chromosome region which encodes a dynamin homolog protein involved in mitochondrial dynamics (72) (MIM #608507). The protein structure is similar to GTPase family members, it consists of two transmembrane anchor domain (TM1 and TM2), two helical hapted repeat regions (HR1 and HR2), a GTPase domain and a very short region in intermembrane space. Both N and C terminal ends are located in the intracellular space [Fig. 6]. Major domains are taxonomically highly conserved (73).

MFN2 has a crucial role in mitochondrial dynamics, especially in mediating fusion, but it is involved in calcium homeostasis, ER-mitochondrion tethering, axonal transport and intrinsic apoptosis as well. These processes are operating from the energy of GTP hydrolysis resulting in the altered structure of protein (74).

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Fig. 6 (A) Gene and (B) protein structure of MFN2 (75, 76). (C) Colors indicate different MFN2 regions where slices represent the number of known mutations in percentages.

Abbreviations: (A) G1-G4 indicate GTP binding domains.

MFN2 is expressed by most cells where mitochondria are present although its dysfunction leads to disease only in the high-energy demanding tissues such as nerve cells. MFN2 is linked foremost to the axonal form of hereditary sensorimotor neuropathies (CMT2A2) but optic atrophy (HMSN-VI) and CNS involvement (HMSN-V) can associate as well (23). MFN2 mostly inherits autosomal dominantly but recessive pattern has been also described (MIM #608507, CMT2A2B). There are more than 110 known pathogenic alterations (77) where phenotypic spectrum, expressivity varies widely (31).

The precise mechanisms are not yet entirely unfolded but more crucial pathways have been identified. Overexpression of MFN2 causes a profound formation of mitochondrial network whereas gene silencing results in extensive fragmentation of mitochondria (78).

In lack of fusion, cell cultures have shown decreased mitochondrial respiration and apoptosis while embryonal lethality and neurodegeneration were observed in animal models (79). MFN2 is also contributing to mitophagy due to PARK2 ubiquitination, PARK2 recruitment and PINK1 phosphorylation which impairments can lead to

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unnecessary apoptosis or necrosis of nerve cells (80). In MFN2 carrier patients’ fibroblast, bioenergetic studies showed mitochondrial coupling defect and an increase of the respiration rate linked to complex II (81).

1.4.2 Less frequent CMT genes

1.4.2.1 EGR2 (Early Growth Response 2) (MIM 129010)

EGR2 gene is localized on 10q21.3 chromosomal region encoding a transcription regulator protein with 3 tandem zinc finger. EGR2 induces the expression of many essential myelin proteins including Cx32, MPZ and MAG where the mutated EGR2 protein has a dominant negative effect on the proteins above, by lowering their overall expression and resulting in neuropathy (82).

Warner et al identified the first heterozygous mutation of EGR2 in CMT. Later, pathogenic alterations were linked to autosomal dominant and recessive forms of DSS and CHN, as well (13, 83). The electrophysiological characteristics are primary demyelination whereas sural nerve biopsy may show a severe loss of myelinated and unmyelinated fibers, onion bulb formation, and focally folded myelin sheaths (13). There are less clinical informations about EGR2 pathogenic alterations, since it proved to be less frequent in CMT population; however, it is commonly associated with severe phenotype indicating its screening especially in cases where early onset (<12 months), rapid progression and premature death (<6 years) is present (84). EGR2 screening should be considered in these cases (13).

1.4.2.2 CTDP1 (C-Terminal Domain of RNA Polymerase II Subunit A, Phosphatase of Subunit 1) (MIM 604927)

The cytogenetic location of CTDP1 is found on 18q23 chromosome region. CTDP1 encodes a general transcription factor which is essential in RNA synthesis. The protein regulates the activity of RNA polymerase II subunit A (POLR2A) with a posttranslational modification. CTDP1 makes possible the initiation of gene expression through POLR2A dephosphorylation (85).

First pathogenic alteration has been described in Rudari Vlax Roma patients with a well- defined syndrome consisted of congenital cataract, facial dysmorphism and neuropathy (CCFDN) (86). CCFDN inherits autosomal recessive and is considered as a founder

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mutation since high carrier rates are present in Roma individuals (~6.9% in Rudari Gypsy, 0.6% in other Roma tribes and 0.0% in non-Roma people) (87, 86). Independently of signs and symptoms, abnormal cell function was observed in each tissue studied (86). In addition to demyelinating neuropathy, skeletal deformities, short stature, congenital nystagmus, cataract, facial dysmorphism, and impaired visus may be present as well.

1.4.2.3 NDRG1 (N-Myc Downstream-Regulated Gene 1)

NDRG1 is localized on 8q.24.22 chromosome region. NDRG1 protein plays a role in cell growth and differentiation as a signaling protein shuttling between the cytoplasm and nucleus and its expression is especially high in peripheral nerve cells, mainly in Schwann cells (88). All the findings indicate the necessity of NDRG1 in nerve cell survival (88).

NRDG1 mutations cause autosomal recessive hereditary sensorimotor neuropathy named as CMT4D or Lom neuropathy after a Bulgarian town. To date, there are only six described mutations where the most frequent p.R148X is considered as a founder mutation in Wallachian Roma patients (89, 88, 90, 91). The disease is characterized by early onset neuropathy (<10 ys), distal paresis and atrophy, sensory impairment including hearing loss, and skeletal deformities (92). Nerve biopsy reveals hypertrophic onion bulbs with partial ensheathment of axons, and ENG shows demyelinating type of neuropathy (93).

1.5 Evaluation of the hereditary neuropathies

Finding the cause of neuropathy is not always easy. A considerable amount of cases stays unsolved after excluding all the treatable disorders. Basically, there are two types of diagnostic approach – pragmatist and completist. Doctors, who are pragmatists, are aiming at minimal possible diagnostic tests to reveal the cause. Other doctors, the completists, try to investigate each possibility, even if it may not have a therapeutic application. These doctors are usually working in tertiary centers as experts of the field (94).

1.5.1 Clinical assessment

The medical history should be always carefully recorded. Clinicians should ask about age of onset, first symptoms, course of progression, family history, possible exposition of

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neurotoxic substances (alcohol, chemotherapeutics, medications, toxins etc.), various infectious diseases (Lyme disease or tick bite, HIV, HCV, HBV) and other medical conditions (malignant neoplasm, lymphoma, multiple myeloma, amyloidosis, autoimmune disease, thyroid dysfunction, trauma, uremia etc.) (1) [Fig. 7]

The routine neurological examination should be performed thorough. To follow the progression, the weakness of muscles (e.g. using MRC scale) and the type and area of sensory involvement should be given exactly by the physician (94). CMT Neuropathy and Examination Scores (CMTNS and CMTES) are powerful and quantitative methods to determine the disease severity and to follow-up the progression. CMTNS consist nine different scores in three groups: symptoms (3), signs (4) and neurophysiology (2). Each score is rated from 0 to 4 regarding the severity (0 – normal, 4 – severe) which means a maximum possible score of 36 points per individual. Severity is ranked as follows: ≤10 mild, 11-20 moderate, >20 severe. CMT examination score can be used as well which exclude the scores of nerve conduction study thus the maximum possible score is 28 points in this case (95). CMT Pediatric Score is designed for children contenting 11 different items with a total score of 44 (96). These measurements are based on Rasch methodology which provides a linear evaluation of progression and follow-up (97). The 6-min walking test is helpful in measuring walking capability and stamina, and prolonged ambulation test can be performed with StepWatchmedical device (98).

Laboratory investigation helps to rule out the acquired causes of polyneuropathy and the following lab parameters are advised to check routinely: serum glucose, calcium (Ca2+), creatinine kinase (CK), lactate dehydrogenase (LDH), thyroid stimulating hormone (TSH), immunoglobulins and B12 vitamin levels, sediment rate; and if it is suspected:

anti-nuclear antibodies (ANA), anti-neutrophil cytoplasmic antibodies (ANCA), ganglioside profile, immunoglobulin electrophoresis, postinfectious panel (Borellia burgerdorfii, human immunodeficiency virus (HIV), hepatitis B and C viruses (HBV and HCV), Clostridium jejuni etc.) or paraneoplastic markers (94).

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Fig 7. Evaluation and differential diagnostic considerations of neuropathies [8].

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26 1.5.2 Nerve conduction study

Nerve conduction study (NCS) is a rapid, non-invasive and cost-effective method to diagnose and follow-up the polyneuropathy. The three different form of neuropathy in CMT are demyelinating, axonal and intermediate types.

Demyelinating neuropathy is characterized by the progressive loss of myelin sheet.

According Kelly electrodiagnostic criteria for predominant demyelination, findings consists at least 3 of the following features (1) prolonged distal latency in ≥2 nerves, (2)

<60% reduction of motor conduction velocity in ≥2 nerves, (3) prolonged latency of F waves as follows: >3ms in arms or >5ms in legs (≥1 nerve), or absence of F-waves in ≥1 nerve (4) partial motor conduction block of ≥1 nerve. The criterion of CIDP also comprises >130% increased temporal dispersion in ≥2 nerves (99, 100).

Axonal polyneuropathy is considered if no definitive signs of demyelination are present.

Kelly electrodiagnostic criteria specify it as follows: (1) >90% of normal NCV if CMAP amplitude is >30% of normal, and >60% of normal NCV if CMAP is <30% of normal, (2) normal or prolonged distal latency in proportion to conduction velocities, (3) normal F waves, (4) no conduction block and (5) fibrillation potentials and neurogenic motor unit changes. Axonal loss has two basic patterns of motor conduction alteration: (1) sparing of few of the fast fibers with severe amplitude reduction and spared MNCV (>50-60 m/s), and (2) sparing only few of slowly conducting fibers which cause a moderate mean nerve conduction velocity (MNCV) (>35 m/s) and prolonged distal motor latency beside the compound muscle action potential (CMAP) amplitude reduction (101).

The definition and criterion of intermediate neuropathy are frequently controversial in the lack of specific electrophysiological protocol. Most recently, Saporta et al considered intermediate neuropathy if MNCV is 38-45 m/s in ulnar nerves and CMAP amplitude reduction is >0.5 mV. However, making a difference between axonal and intermediate types is sometimes hard due to the sparing only of slowly conduction fibers in axonal neuropathy (18).

Certain genes are specific for distinct types of neuropathy but many cannot be unambiguously categorized (e.g. GDAP1, GJB1, MPZ etc.). In special cases, uncommon

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signs can be also present such as severe temporal dispersion (CMTX1) or conduction blocks (HNPP) which may implicate difficulties in differential diagnostic follow-up.

1.5.3 Nerve biopsy and imaging studies

In the first part of 20th century, nerve biopsy and histology meant to be the best diagnostic approach of CMT. In demyelinating form, moderate to severe reduction in density of myelinated fibers, hypermyelination or demyelination, and a high number of onion bulb formations can be observed. The axonal form is characteristic of lack, loss, or preservation of nerve fibers and signs of regeneration. After identification some of the causative genes, clear genotype-phenotype correlations have been found with certain histological changes such as focal axonal alterations due to MFN2 and NEFL mutations, congenital amyelination in SOX10, EGR2, CTDP1 alterations or tomacula in HNPP (23).

In the genetic era, nerve biopsy is mainly obsoleted and is required and justified only in few cases like suspected diagnosis of CIPD, amyloidosis or small fiber involvement (23).

Nerve ultrasound is widely used as a differential diagnostic application in evaluation of neuropathies. Lately, more studies attempted to describe the characterization of different CMT subtypes like CMT1A or CMT1B. Measurement of cross-sectional area (CSA) of peripheral nerves was significantly increased in CMT1A, and CSA correlated with the disease severity (CMTNS) and peripheral nerve function (102). In CMT1B, increased CSA of median and cranial nerves was observed (103). Axonal neuropathy is not characteristic for increased CSA based on several cases (102).

MRI neurography in CMT may reveal hypertrophic roots typically with onion bulb sign which represent hypertrophic demyelination (CMT1 and CMT4) and depicting also nerve entrapment or impingement occasionally (104-106). In certain cases, some enhancement in nerves may be also seen but it is infrequently a prominent feature. Quantity muscle MRI based on Dixon sequences and quantitative muscle ultrasound is suitable to measure precisely and reliably the thigh and leg muscle atrophy as well as denervation-related fatty substitution which is held as the most responsive measure available in the monitoring of CMT progression (107, 24, 108).

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28 1.5.4 Genetic diagnostics

The development and decreasing price of molecular genetic methodologies have opened new frontiers in diagnostics of hereditary disorders. Genetic diagnosis can likely prove CMT since positive test confirms hereditary neuropathies even in uncertain cases;

however, negative test results do not exclude CMT. In the last decade, numerous study attempt to define an ultimate strategy of genetic testing but these were partly unsuccessful. Each population showed divergent gene frequencies and numerous factors biased its universal application so these should be considered more likely as a rule of thumbs [Fig. 8] (51, 27).

Recently, new generation sequencing (NGS) lifted to a new level of diagnostic perspectives in hereditary disorders. Specific CMT panels may reveal other possible genetic modification factors which potentially contribute in the different biological pathway and modulate the penetrance and/or the expressivity of the overall phenotype.

However, the absolute cost of NGS is still high and the quality and reliability of results are occasionally questionable. Even today, Sanger sequencing and MLPA are held the gold standards in SNV’s and CNV’s analyses despite their limited output but high fidelity and feasibility (109).

In regard to the inheritance, the four most common genes can be advised for analyzing before any more extensive screening: in CMT1 PMP22, GJB1, and MPZ, and in CMT2 GJB1, MFN2, and MPZ in order, based on the following scheme (51).

Fig. 8. A decision tree of successive analysis of suggested genes in CMT (51).

no male to male transmission

no male to male transmission

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29 1.6 Management of hereditary neuropathies

1.6.1 Genetic counseling

Since the patients’ genetic diagnosis is irrevocable during his/her entire life, genetic counseling has an enormous liability in the management of hereditary disorders such as CMT. The process of genetic counseling is dedicated to inform the patient about the purpose and possible outcomes of genetic testing just as about the expected psychological and physical burdens of the diagnosis and how can it affect the offsprings and other relatives (110).

The Hungarian Parliament enacted the state law about human genetics in 2008 (2008. évi XXI. törvény) which determine the major points of genetic counseling, testing, and research. Even so, in absence of implanting regulations (31.§), some crucial details of its execution are still undefined thus fundamental medical consensus and best practice should be supreme in these cases.

Major points of genetic counseling by the law:

 A patient, who has the capacity, owns the right for a private counseling.

 Patients must be informed about the purpose of testing, potential consequences, methods of processing and storing the genetic material, and data protection, and possible benefits and disadvantages of refusal, (6.§ (2)).

 Genetic counseling is necessary before and after a genetic testing (6.§ (2)).

 Prognosis, therapeutic possibilities must be communicated and psychological support must be offered in appropriate cases (6§ (4)).

 Patients have the freedom to know or not to know the genetic result (6.§ (7)).

 The genetic result must not be shared with a third party unless in possession of a permission document representing conclusive evidence, or in a case of potentially affected family members with clinical relevance (conception, prevention, therapy, etc.) related to the result (7.§)

 Patients must give their written informed consent before sample is taken (8.§) Although Charcot-Marie-Tooth disease does not affect the life expectancy in general, presymptomatic counseling and testing require one or more sessions (predecision and

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pretest counseling). Here, the clinicians need to be more prudential regarding the potential impact of diagnosis, benefits, and disadvantages. The freedom to not know of the genetic result must be kept in sight. Every case is unique and requires experience in leading the patient to the right choice. The councelor has to evaluate the possible impact and may evade the telling of diagnosis but always the patient decides finally.

Very important that presymptomatic screening is strictly forbidden and unethical under the age of legal competence unless its necessity proved vital regarding a close relative (111).

Another relevant question is the person of the counselor. In absence of implanting regulations, the law does not specify who can provide genetic counseling. Reckon with the fundamental principle of the law, best practice and medical ethics, no other professional should give a genetic counseling but a trained medical doctor with extensive knowledge of the field, otherwise the right to information (6.§ (2)) can be easily compromised.

1.6.2 Therapy and treatment

Until now, no effective treatment was developed in CMT although there are some efforts to moderate the overall disease burden. It is very important staying active and keeping up the strength with physiotherapy or regular exercises (112). Occupational therapy can help in coping with daily tasks (113). Orthoses, orthopedic shoes or canes can support the walking and orthopedic surgeries can correct the feet deformities and Achilles contractures if needed (114).

Many promising compounds are in different phases of drug research. PXT3003, which is a combination of naltrexone, baclofen, and sorbitol, is currently in phase III (NCT02579759). The encouraging results of the animal model showed its beneficial effect in lowering PMP22 overexpression (115). Downregulation of PMP22 with progesterone antagonist (ulipristal-acetate) is right in phase II (NCT02600286) (116).

There are novel efforts as well aiming the gene silencing of PMP22 by lowering its expression with antisense oligonucleotides and small interfering and hairpin RNAs (117).

Gene therapy with intrathecal lentiviral vectors is also in scope in CMTX1 treatment since

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stable Cx32 proteins were produced and maintained in treated Gjb1-knocked out mice (118). L-serine supplementation is promising in SPTLC1-2 defects (NCT1733407) (119).

1.6.3 Avoid of medications

There is little information about drugs which should be ultimately avoided.

Unambiguously, neurotoxic medication can progress the neuropathy so the application of these drugs should not be used in general. There is a clear evidence that administration of vinca alkaloids and taxols may cause rapid and severe progression and nerve injury in CMT patients even in mild or asymptomatic CMT (120). In higher percentages of cases, nitrous oxide (50%), metronidazole (23%), nitrofurantoin (20%), phenytoin (11%), statins (10%) and sertraline (9.5%) can initiate an exacerbation of neuropathy. Other drugs have moderate to doubtful risk and should be considered the treatment individually and assess the improvement or worsening of disease helping in decision whether benefits surpass risks (121).

1.6.4 CMT and pregnancy

There are only a few publications about pregnancy risks and CMT and results are controversial. A publication from 2012 reviewed the natural history of pregnant women with neuromuscular disorders (NMD), including CMT. The study concluded that the worsening of status was present in 31% which did not remit in 22% of patients. Very important that CMT did not influence significantly pregnancy outcome, labor and delivery, mode of delivery, preterm birth and neonatal outcome (122). Another study found that pregnancy with CMT indicated a higher occurrence risk of presentation anomalies and bleeding postpartum. The rate of caesarean section was doubled and forceps was used three times as often in the CMT group as in individuals without NMD.

Most of the CMT operative deliveries were emergency sections (123).

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2. Objectives

In the last decades, the successive exploration of CMT made the diagnostics more successful and exact than ever. Furthermore, numerous studies proved that a well- characterized population and gene specific epidemiological data can enhance the efficiency and lower the costs of diagnostics. In this study, we aimed to investigate Hungarian CMT patients, unravel the genetic cause of the disease even in still unsolved cases and assess the phenotypical variability and spectrum of different causative genes.

The aims were the followings:

1. To estimate the frequency of most common neuropathy genes – PMP22, GJB1, MPZ, MFN2, EGR2, CTDP1 and NDRG1 – in an extensive cohort of Hungarian CMT patients.

2. To assess the disease features and atypical signs and symptoms of CMT in this cohort and detailed descriptions of the phenotype of novel pathogenic and likely pathogenic alterations.

3. To highlight various genotype-phenotype correlations between different CMT subgroups, especially between clinically well-characterized female and male CMTX1 patients.

4. To analyze patient for rare variants with high- throughputgenetic methods and identify the causative gene.

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3. Methods

3.1 Clinical and electrophysiological characterization of the cohort studied

531 Hungarian CMT patients were enrolled (242 females and 289 males; mean age of 39.3±17.6, CI95% (37.78 to 40.76)). All individuals were born in Hungary, whereof 55 patients (10.4%, CI95% (0.78-0.13)) were likely of Roma origin. Some of the novel alterations were tested in 350 healthy control individuals (209 female, 141 male; mean age 39.88±14.87; CI95% (38.32 to 41.44)) as well. All individuals were born in Hungary and descended from Hungarian ancestors. Written informed consent was obtained from all individuals. Molecular genetic analysis was performed for diagnostic purposes in all investigated patients. The study was approved by the Ethical Committee of Semmelweis University (119/PI/12, 7891/2012/EKU).

Patients routinely underwent neurological examination. Age of onset and family history was taken in all cases by asking about other affected relative and first neuropathy related symptoms, respectively. Distal hereditary motor neuropathy (dHMN) and hereditary sensory and autonomic neuropathy (HSAN) were excluded as well.

Nerve conduction studies (NCS) were performed by standard techniques (Dantech Keypoint, Denmark) with superficial registration and stimulation of sensory, motor and mixed nerves. Routinely investigated nerves: sural sensory, peroneal and tibial motor nerve conduction and F-waves, ulnar and median nerves sensory and motor conduction inclusive F-waves. Demyelinating neuropathy was diagnosed if the distal latency and/or F-latency was prolonged and/or the conduction velocity was reduced. The increased temporal dispersion was taken as a sign of demyelination as well. Focal conduction block was ruled out. Diffuse amplitude-reduction and no evidence of demyelination indicated an axonal loss. Nerve lesion was diagnosed as intermediate type if amplitude-reduction was present with decreased nerve conduction velocity but the criteria of primary demyelination have not been fulfilled (124). We also categorized patients based on nerve conduction velocity exclusively, as follows: NCV of ≤38m/s indicated CMT1 while NCV of >38m/s indicated CMT2. Normal values in reference to patients’ height and gender were calculated using the Dantec Keypoint Software (Keypoint Software v.3.03).

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Patients were considered to suffer from CMT if the clinical and electrophysiological signs of motor and sensory neuropathy were present and the family history revealed other affected family members. First and second degree relatives with similar characteristics of neuropathy were considered to carry the same pathogenic variant. In sporadic cases, the causes of acquired neuropathy (e.g. metabolic, toxic, inflammatory, infectious and tumor associated polyneuropathies) were excluded using extensive differential diagnostic workup. Further diagnostic tests or procedures (e.g. additional laboratory investigation, audiological evaluation, brain and/or spinal MRI or CSF analysis) were performed if required based on the clinical picture. The severity of the disorder was assessed using the CMT examination and/or neuropathy score part of this retrospectively (125).(124)(123).

3.2 Genetic testing

DNA was isolated from whole blood using the QIAamp DNA mini kit (QIAGEN®).

Quantitative changes in the PMP22 gene was analyzed with multiplex ligation-dependent probe amplification assay (MLPA) (SALSA MLPA 33 CMT1 probemix, MRC Holland).

Copy number variation of the GJB1 gene was screened by real-time PCR methodology with SYBR Green (ThermoFisher®) staining. Copy number was determined using the ddCt method and compared to human serum albumin. The total coding region of GJB1 (ENST00000374022, NM_001097642), MPZ (ENST00000533357, NM_000530), EGR2 (ENST00000242480, NM_000399), MFN2 (ENST00000235329, NM_014874), PMP22 (ENST00000395938, NM_153321) were analyzed using Sanger sequencing with specific primers (Suppl. 3) and compared to the human reference genome using NCBI’s Blast® application. Hotspot mutations in CTDP1 and NDRG1 (TaqI) genes were tested with the PCR-RFLP (CTDP1-NlaIII, NDRG1-Taq1) methodology.

CMT1 patients were first screened for PMP22 duplication. If this was negative, successive analysis of the coding region of GJB1, MPZ, EGR2, and PMP22 was performed. In CMT2 cases, the sequence of GJB1 was first analyzed followed by MFN2 and MPZ genes. In both CMT1 and CMT2 patients, GJB1 deletion analysis was also performed if other tests did not identify the causative gene. GJB1 was only tested if male to male transmission was absent. Patients with likely Roma origin were screened for founder mutations as well.

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In control cohort novel alterations were also screened with PCR-RFLP: GJB1: c.38 T>A - Hpy166II (New England Biolabs® R0616S); c.557 A>G - NciI (New England Biolabs®

R0196S); c.582 G>C – BmtI (New England Biolabs® R0658S))

Exome Capture was performed in Miami University, Hussman Institute. The procedure was executed according to the manufacturer's protocol. For genomic DNA library preparation, TruSeq® DNA Sample Prep Kit v2‐Set A (Illumina) and NimbleGen SeqCap EZ Human Exome Library v3.0 Kit exome enrichment (Roche) was used. Crude pre‐captured genomic library was analyzed by Agilent 2100 Bioanalyzer 1000 DNA chip to assess library quality. It was followed by exome enrichment, preparation of hybridized libraries, purification and library quality and quantitative control of qPCR. All library pools were sequenced on the HiScanSQ Illumina sequencing platform, using 2 × 95‐bp pair‐end sequencing protocol, with an extra 9‐bp index sequencing run. 95‐bp paired‐

reads were aligned to the human reference genome (hg19). The alignment was executed with Burrows–Wheeler aligner (BWA) software. For variation calling, Samtools software was used. We screened genes which are associated with neuropathies and likely involved in homeostasis of peripheral neurons. This gene set was compilated based on UNIPROT, NextProt, OMIM, and NCBI databases (Suppl. 3.). The variants were checked in Clinvar, dbSNP and Ensembl database. We focused on non‐synonymous variants, splice acceptor and donor site mutations, and short, frame shift coding insertions or deletions (indel).

Exonic frameshift and stop mutations were considered as damaging. Missense mutations were prioritized, which was based on the protein prediction score annotations given by Polyphen2, SIFT, MutationTaster and GERP software (SIFT < 0.1, Polyphen2 >0.5 > 3, MutationTaster: disease causing). Using ACMG guideline, the pathogenic and likely pathogenic variants were confirmed by Sanger sequencing.

Target sequencing of patients with predominant motor function impairment was performed using Illumina MiSeq platform and an in-house compiled Agilent Haloplex capture panel of following genes: AARS, AIF1, ALS2, ANG, ASAH1, ATP7A, BICD2, BSCL2, C9ORF72, CHCHD10, CHMP2B, DCTN1, DNAJB2, DNMT1, DYNC1H1, EXOSC3, EXOSC8, FBXO38, FIG4, FUS, GAN, GARS, GEMIN2, GLE1, HEXA, HEXB, HINT1, HSPB1, HSPB3, HSPB8, IGHMBP2, LAS1L, LMNA, MAPT, MEGF10, MT3, NAIP, NEFH, OPTN, PFN1, PLEKHG5, PNPLA6, PRDX3, REEP1, SCO2, SETX, SIGMAR1, SLC52A2, SLC52A3, SLC5A7, SMNDC1, SOD1, STMN1, TARDBP, TFG,

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TRPV4, UBA1, UBQLN2, VAPB, VCP, VIM, VRK1. Bioinformatic algorithm, variant calling and variant analys was identical to the exome capture.

3.3 In silico, pathogenicity and statistical analyzes

In silico analyses were performed with PolyPhen2, MutationTaster and SIFT softwares.

The significance of detected alterations was checked with HGMD (www.hgmd.cf.ac.uk), dbSNP (www.ncbi.nlm.nih.gov/SNP/), ClinVar (www.ncbi.nlm.nih.gov/clinvar/) and CMT database (http://www.molgen.ua.ac.be). The nature of novel alterations was assessed based on the ACMG guideline.

The group comparisons were performed with independent sample t-test and Mann Whitney U test regarding means. Percentages were compared with Chi square test. p values of <0.05 was considered statistically significant. Odds ratio (OR) in case control studies and the 95% confidence intervals (CI95%) for proportions and means were calculated using standard formulas.

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37 4. Results

4.1 Clinical and electrophysiological assessment

From the 531 studied CMT patients, 409 (77.0% (CI95% (0.734 to 0.805)) were classified as CMT1 and 122 (23.0% (CI95% (0.194 to 0.265)) as CMT2. Family history was positive in 148 cases (51.0% CI95% (0.453 to 0.568)) while 142 patients (49.0% CI95%

(0.433 to 0.548)) were sporadic. The inheritance pattern was autosomal dominant in 123 cases with a mean patient per family ratio (P/F) of 2.6. X-linked dominant and autosomal recessive inheritance were present in 13 (P/F: 3.4) and 12 (P/F: 1.8) cases, respectively.

CMTES and CMT related additional features could be assessed in 309 cases (58.2%

CI95% (0.540-0.624)). The mean CMT examination score was 8.9±4.3 (CI95% (8.42- 9.38), with a minimum of 0 and a maximum of 22. Symptoms began before the age of 30 in 69.3% of CMT cases; however, the age of onset ranged between the first and seventh decade of life. Additional features were found in a total of 22.3% of patients (CI95%

(0.177-0.270)) as follows: CNS involvement in 7.8% (24), facial, glossopharyngeal and recurrent laryngeal nerve palsy in 5.2% (9, 4 and 3 respectively), bilateral sensorineural hearing impairment in 4.9% (15) (<40 dB and age at onset <40 years), immune dysfunction in 2.9% (9), autonomic nervous system (ANS) involvement in 1.6% (5), cataract in 1.3% (4) and optic atrophy in 0.7% (2) of the cases.

4.2 Genetic testing and distribution of genetic subtypes

Within the studied cohort, genetic testing confirmed the causative gene in 276 CMT1 (67.2% CI95% (0.617 to 0.728)) and in 42 CMT2 (34.4% CI95% (0.260 to 0.428)) patients. Altogether 318 CMT patients (59.9% CI95% (0.557 to 0.641)) received a genetic diagnosis, while in 213 individuals (40.1% CI95% (0.359 to 0.443)) we could not detect any pathogenic alterations within the genes studied.

Regarding the entire CMT cohort, the most frequent causative gene alteration occurred in PMP22 (40.1%) followed by GJB1 (9.6%), MPZ (4.5%), MFN2 (2.4%) NDRG1 (1.5%), EGR2 (0.8%) and CTDP1 genes (0.8%) [Table 2]. Homozygous founder mutations in NDRG1 and CTDP1 genes were present in 21.8% of investigated Roma patients, with eight and four cases respectively.

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Table 2 – Pathogenic and likely pathogenic variants identified in the studied cohort and electrophysiological features, age of onset and disease severity of probands. Abbr.: p.s. – present study

Gene Alteration AA

change Significance Proband

/patients NCV Gender of probands

Probands’

age on onset (decade)

Probands’

disease severity

Ref

PMP22 dupl. - pathogenic 136/212 CMT1 male/female first to fifth

mild to

severe (126) PMP22 c.353 C>T T118M pathogenic 1/2 CMT1 male second mild (127, 128) GJB1 c.38 T>A V13E likely

pathogenic 1/2 CMT2 male second severe (129)

GJB1 c.43 C>T R15W pathogenic 1/1 CMT1 male first moderate (130) GJB1 c.187 G>A V63I pathogenic 1/2 CMT2 male second moderate (131) GJB1 c.224 G>A R75Q pathogenic 2/5 CMT1 male/female second moderate (132) GJB1 c.265 C>G L89V pathogenic 1/2 CMT1 male second severe (133) GJB1 c.287 C>G A96G pathogenic 2/7 CMT1/

CMT2 male/female third and

fifth moderate (134) GJB1 c.319 C>T R107W pathogenic 1/1 CMT1 female third mild (135) GJB1 c.379

A>G I127V pathogenic 1/3 CMT1 male second moderate p.s.

GJB1 c.425 G>A R142Q pathogenic 1/2 CMT1 male first severe (136) GJB1 c.490 C>T R164W pathogenic 1/1 CMT1 female first mild (137) GJB1 c.491 G>A R164Q pathogenic 1/3 CMT1 male first severe (138) GJB1 c.514 C>G P172A pathogenic 1/4 CMT2 male second severe (139) GJB1* c.557 A>G E186G likely

pathogenic 1/4 CMT2 male third moderate (129) GJB1* c.582 G>A M194I likely

pathogenic 1/3 CMT1 male second mild (129)

GJB1 c.614 A>G N205S pathogenic 1/3 CMT1 male first moderate (136) GJB1 c.623 G>A E208K pathogenic 1/2 CMT1 male second mild (140) GJB1 c.712C>T R238C pathogenic 1/2 CMT1 male first moderate (141) GJB1 deletion - pathogenic 2/4 CMT1/

CMT2 males second moderate (142)

Ábra

Fig. 1 A: The diagram indicates the extension of the literature related to Charcot-Marie- Charcot-Marie-Tooth  neuropathies  in  the  previous  decades
Table 1. The different classifications of hereditary motor and sensory neuropathies since  the  first  descriptions
Fig. 2 27 -year-old male patient harboring the entire deletion of the coding region of  GJB1 gene
Fig. 3 Functions of proteins encoded by different CMT associated genes (23).
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